This project is focused on the structure, function, and conformation of an integral membrane proton pump, transhydrogenase. Transhydrogenase (TH) is an essential enzyme of the respiratory system in mitochondria and bacteria. Proton translocation through membrane-intercalated domain II is coupled to hydride transfer between NAD(H) and NADP(H) bound to extramembranous domains I and III. In the forward direction, the proton motive force shifts the equilibrium toward NADPH formation; in the reverse direction, transhydrogenation from NADPH to NAD results in creation of a proton gradient. The structure of the intact enzyme is unknown, and the mechanism for coupling binding energy and conformational change with proton translocation is unsolved. Structural and functional experiments will develop a picture of this complex molecular machine and its catalytic mechanism. Three Aims address key aspects of TH function: the sites of proton uptake and release in domain Ill, the mechanism of hydride transfer between domains I and III, and the structure and conformation of the intact enzyme. A fourth Aim addresses the mechanism by applying mutagenesis and functional assays to evaluate the coupling of proton transfer in domain II with conformational and binding events in domain III. The experiments entail protein expression and purification, mutagenesis, enzymatic activity assays, preparation of monodisperse, detergent-solubilized TH, negative stain electron microscopy, crystallization in lipid bilayers, single-particle and 2D crystal electron cryo-microscopy, crystallization of detergent-solubilized TH, NMR of expressed domain III, and x-ray crystallography of expressed domains I and III, individually and in cocrystals. [unreadable] [unreadable]